Development of Variants of a Commercial Reverse Transcriptase with Enhanced Thermal Stability

• Grant number: 24/05367-4
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: February 01, 2025
• End date: October 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Felipe Ribaldo Ferreira da Costa
• Grantee: Felipe Ribaldo Ferreira da Costa
• Company: Cellco Biotec do Brasil Ltda
• CNAE: Fabricação de produtos químicos não especificados anteriormente ; Pesquisa e desenvolvimento experimental em ciências físicas e naturais
• City: São Carlos
• Associated researchers: Amanda Bernardes Muniz ; Naiara Utimura Torres
• Associated scholarship(s): 25/03075-9 - Development of variants of a commercial reverse transcriptase with enhanced thermal stability, BP.PIPE

Abstract

Reverse transcriptases (RTs) are enzymes that synthesizes a complementary DNA strand from an RNA molecule as a template. Routinely used in Molecular Biology laboratories, RTs are largely used in the fields of health, agriculture, veterinary and industry. However, a recurring challenge to RTs is the manipulation of long RNAs and/or those with complex secondary structures that affect the synthesis, leading to incomplete products. Alternatively, using high reaction temperatures is a recommended strategy to enable or optimize reverse transcription. Therefore, ideal RTs for biotechnological applications are thermostable, synthesizes long chains of complementary DNA and have satisfactory activity at high temperatures. In this context, several genetic engineering methodologies were used to improve their properties, highlighting the Moloney Murine Leukemia Virus RT mutants (M-MLV-RT). The Brazilian market still shows significant dependence on RT imports, resulting in high costs and long delivery times. Thus, searching for improved variants of M-MLV-RT compatible with RTs of international competitors is a promising strategy. The project proposed by Cellco Biotec seeks to supply the scientific and experimental demands of the domestic market with an innovative and genuinely Brazilian RT, of high quality and with economic advantages - fair and competitive prices, agility and prompt availability. To achieve this, random mutations will be introduced by epPCR into the coding sequence (CDS) of a genetically engineered version of the M-MLV-RT already commercialized by the company. The amplified products will be cloned in E. coli expression plasmids and the generated mutants will be screened by the Hot Colony Filtration Blot method to select thermostable proteins at the colony level. Additionally, point mutations in the D200, L603 and E607 residues will also be induced by site-directed mutagenesis to the control CDS and their effects will be evaluated. Therefore, this proposal has the potential to reach level 8 of technological maturity at the end of its plausible Phase 2, obtaining an improved mutant RT with eminent commercial and scientific return to the national reality. (AU)